Waveguide immunosensor with coating chemistry providing enhanced sensitivity

ABSTRACT

Methods and apparatus for evanescent light fluoroimmunoassays are disclosed. The apparatus employs a planar waveguide and optionally has multi-well features and improved evanescent field intensity. The preferred biosensor and assay method have the capture molecules immobilized to the waveguide surface by site-specific coupling chemistry. Additionally, the coatings used to immobilize the capture molecules provide reduced non-specific protein adsorption.

BACKGROUND OF THE INVENTION

1. Field

This application relates to the art of analyzing samples for particularsubstances by means of fluorescent binding assays, and more particularlyto apparatus, compositions and methods for such assays employingevanescent light.

2. State of the Art

Biosensor apparatus based on optical detection of analytes byfluorescence of tracer molecules, have attracted increasing attention inrecent years. Such apparatus are useful for both diagnostic and researchpurposes. In particular, biosensors for a solid-phase fluoroimmunoassay,are becoming an important class of optical biosensor. A technique knownas TIRF or total internal reflection, is one method forexcitation/detection of fluorescence useful with biosensors forsolid-phase assays.

In a typical such apparatus the biosensor is an optical substrate suchas a fiber optic rod, to which is adsorbed or covalently bound a bindingagent specific for a desired analyte. When the sensor is irradiated withlight of an appropriate wavelength, the binding of the analyte to theimmobilized binding agent results in a change (either a decrease orincrease) in fluorescent emission of a tracer substance. The tracersubstance may be the binding agent, the analyte, the complex, or athird, added tracer molecule.

It is desirable to have a system in which the desired sensitivity isachieved without requiring a "wash" step to remove unbound tracer beforethe fluorescence is measured. One approach to such a system has been toutilize evanescent light to selectively excite tracer molecules directlyor indirectly bound to the immobilized binding agent. Evanescent lightis light produced when a light beam traveling in a waveguide is totallyinternally reflected at the interface between the waveguide and asurrounding medium having a lower refractive index. A portion of theinternally reflected light penetrates into the surrounding medium andconstitutes the evanescent light field. The intensity of evanescentlight drops off exponentially with distance from the waveguide surface.

In fluorescence assays using evanescent light, the waveguide is usuallyglass or a similar silica-based material, and the surrounding medium isan aqueous solution. The region of effective excitation by evanescentlight in that situation is generally from about 1000 to about 2000 Å(angstroms). This region is sufficient to include most of the tracermolecules bound to the waveguide surface by means of interaction betweenthe capture molecules and the analyte. However, the bulk of the unboundtracer molecules remaining in solution, will be outside the range ofeffective excitation and thus will not be stimulated to emitfluorescence.

Desirably, an immunofluorescent biosensor should be capable of detectinganalyte molecules at concentrations of 10⁻¹² or below. To date, mostreports of evanescent-type biosensors indicate that at best,concentrations of 10⁻¹¹ could be detected.

It is further desirable for speed and convenience in "routine" testing,for example testing of blood bank samples for viral antibodies, to havean evanescent immunofluroescent biosensor which is disposable and whichprovides multi-sample measurement capability. Multi-sample capabilitywould allow a test sample and a control sample (such as a blank, or, fora competition-type assay, a sample preloaded with tracer molecules) tobe simultaneously illuminated and measured. Simultaneous multi-samplecapability would also speed up the process of analyzing multiple samplesand would reduce the effects of variation in the level of exciting lightwhich are known to occur with typical light sources. However, in atypical prior art evanescent light device such as that of Block et al,U.S. Pat. No. 4,447,546, the waveguide is a fiber optic rod whose shapemakes it difficult to build a multi-well biosensor.

Another factor which affects the attainable sensitivity relates to theintensity of excitation light emitted from the waveguide. The intensityof fluorescence emitted by tracer molecules is in part dependent on theintensity of exciting light (which is the evanescent field). Therefore,increased evanescent light intensity should provide increasedfluorescence which in turn would improve the detection sensitivity. Thelevel of evanescent light is in turn dependent on the intensity of thelight beam propagating in the waveguide and on the efficiency ofreflection of light at the interface between the waveguide and thesurrounding medium. The typical rod-shaped waveguides are not aseffective in internal reflection as a design having two parallelsurfaces would be.

Previous methods of immobilizing antibodies to optical substrates inevanescent biosensors also present some problems causing reduction insensitivity. Many such methods utilize the ε-amino groups of lysineresidues in the protein. This approach has at least two significantdisadvantages due to the fact that most proteins have multiple lysineresidues. First, the presence of multiple potential coupling sites(multiple lysine residues) results in multiple random orientations ofantibodies on the substrate surface. If the substrate-coupled lysineresidue is near the N-terminal of the antibody molecule, the antibody'santigen binding site (which is near the N-terminal) may be effectivelyunavailable for binding of the analyte.

Second, if multiple lysines on the same antibody molecule are coupled tothe substrate, the molecule may be subjected to conformational strainswhich distort the antigen binding site and alter its binding efficiency.For capture molecules immobilized by typical prior methods, generallyonly 20% or less of the binding sites are functional for analytebinding. Thus, it is desirable to have a site-specific method forcoupling of the antibodies or other proteins, so that the capturemolecules will be uniformly oriented and available for analyte binding.

Another problem relates to the levels of non-specific binding to theantibody-coated surface of the optical substrate. These levels are oftensufficiently high to make detection of analyte at concentrations belowabout 10⁻¹⁰ molar (abbreviated M) very difficult. Non-specific bindingcan be reduced by including a wash step after the sample is incubatedwith the coated substrate, to remove unbound tracer molecules. However,this is time-consuming and complicates the assay procedure. Forconvenience, a one-shot or homogeneous assay, that is, one which doesnot require a wash step, is much to be preferred. Second, non-specificbinding can be a serious problem unless the surface is "passivated" witha masking agent such as bovine serum albumin or with a thin coating ofhydrophilic polymer such as poly(ethylene glycol) or poly(methacrylate).Without such passivation (which introduces yet another step into theprocedure), non-specific binding can be 50% or more of the specificbinding. Even with passivated surfaces, non-specific binding can besufficient to reduce detection sensitivity and reproducibility.

Thus, a need remains for an evanescent biosensor apparatus with improvedsensitivity for detection of analytes at picomolar concentrations andbelow. A need further remains for an immunofluorescent assay andbiosensor with properties of low non-specific binding and havinguniformly oriented capture molecules.

SUMMARY OF THE INVENTION

The invention comprises a system including both apparatus and methodsfor an evanescent-light immunofluorescence assay capable of detectingsub-picomolar concentrations of analytes in solution.

The apparatus includes a biosensor comprising a planar waveguide havinga plurality of immobilized capture molecules on at least one surface,the capture molecules being constructed to selectively bind a desiredanalyte. The waveguide surface forms one wall of at least one samplereservoir for holding a sample solution. A light source is included inthe apparatus and operably arranged to focus light into the waveguide,where internal reflection within the waveguide results in the productionof an evanescent light field which penetrates into the sample solution.In the assay, the test solution also contains a plurality of tracermolecules constructed to emit fluorescence upon stimulation by theevanescent light. The apparatus further includes detection means fordetecting fluorescence emitted by the tracer molecules, which isreflective of the amount of analyte bound to the capture molecules.

In a preferred embodiment, multiple wells or channels are provided onthe surface of the waveguide, to permit simultaneous comparison offluorescence from control and sample solutions.

In another preferred embodiment, a substantial portion of thesurrounding edge of the waveguide is coated with a reflective coating toprevent light from escaping through the edge, thereby increasing theintensity of the evanescent field.

In another preferred embodiment, the biosensor has the capture moleculessite-specifically immobilized such that the percentage of capture sitesavailable is 50 to 75% or more of the number of immobilized capturemolecules. In a further preferred embodiment, the waveguide coatingsused to immobilize the capture molecules are selected to be resistant tonon-specific protein binding.

The invention includes a method of immobilizing the capture molecules ata selected site on the molecule, so that the immobilized capturemolecules are substantially uniformly oriented on the waveguide surface.In the method the waveguide surface is coated with a first coatinghaving selected available reactive groups, and the capture molecules tobe immobilized are treated to modify a single moiety on each capturemolecule to produce activated capture molecules. The modified moiety isconstructed to bind to the reactive groups of the first coating. Thecoated surface with the activated capture molecules under conditions tocause the modified moiety to couple to the first coating and therebyimmobilize the activated capture molecules to the waveguide surface.

In one procedure, the waveguide is coated with avidin, and the capturemolecules are conjugated to a biotin moiety which has a very strongaffinity for avidin. In another procedure, the waveguide coating is ahydrogel film formed of polymethacryloylhydrazide treated to producefree maleimido groups, and the Fab' capture molecules are oxidized toproduce reactive thiol groups which can then be reacted with themaleimido groups. In a third embodiment, a silanized waveguide surfaceis further coated with polyethylene glycol derivatized withethylenediamine groups. These groups are then reacted with oxidized Fab'capture molecules.

A particular embodiment of the invention is sandwich-type assay capableof detecting sub-picomolar concentrations of human chorionicgonadotrophin. However, the coupling chemistry and apparatus describedare useful for any type of fluoroimmunoassay for which the necessarybinding reagents are available, including tests for serum antibodies toselected pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a fluorescent immunoassay apparatus ofthe invention;

FIG. 2 is a side view of a portion of the waveguide and the biochemicalcomponents of a competition immunofluorescent assay according to theinvention;

FIG. 3A is a top view of a flow biosensor of the apparatus of FIG. 1;

FIG. 3B is a side cross-section view of the flow biosensor taken alongline B--B in FIG. 3A;

FIG. 3C shows the waveguide in isolation as it could be arranged withrespect to a cylindrical lens and incoming and reflected light waves;

FIG. 4A is an elevational view of a two-channel flow biosensor of FIGS.3A-3B with respect to exciting light beams and the collection offluorescence in an immunoassay;

FIG. 4B is a schematic diagram of the two-channel biosensor indicatingthe arrangement of two types of detection devices, a CCD detector and aspectrometer slit, with respect to the waveguide regions;

FIG. 4C is a diagram of fluorescence intensities as they might bedetected from the two channels of a biosensor arranged according toFIGS. 4A and 4B;

FIG. 5A is an elevational view of an alternate embodiment of amulti-channel biosensor;

FIG. 5B is a side view of the biosensor of FIG. 5A;

FIG. 5C is a side view of the biosensor of FIG. 5A in a verticalorientation with a sample solution therein;

FIG. 5D is a cross-sectional view of the biosensor taken along line D--Din FIG. 5C;

FIG. 6 is an elevational view of an alternate embodiment of a multiwellbiosensor;

FIG. 7A is a chart depicting fluorescence intensity data from a sandwichfluoroimmunoassay for detecting an antibody, and performed with theapparatus of FIG. 1 according to a first assay format;

FIG. 7B is a chart depicting data from a sandwich fluoroimmunoassayperformed with the apparatus of FIG. 1 according to an alternate assayformat;

FIG. 8 is a chart comparing the fluorescence enhancement observed withthe assay formats of FIGS. 7A and 7B;

FIGS. 9A-F are charts depicting data from an alternate scheme for asandwich fluoroimmunoassay for detecting an analyte using acorresponding antibody;

FIGS. 10A-D are charts depicting data from a displacementfluoroimmunoassay performed with the apparatus.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

A light source 100 provides a light beam 102 which is directed by meansof mirrors 104, 106, 108 to an optical biosensor indicated generally at120 (FIG. 1). In the working embodiment, light source 100 is an argonlaser capable of emitting light at wavelengths of between about 488 and514.5 nanometers (abbreviated nm). In an alternate embodiment, a laserdiode emitting at wavelengths of 600 to about 700 nm can be used aslight source 100. Depending on the requirements of the fluorescenttracer, light source 100 may also be embodied as any other laser orother high-intensity light source emitting a sufficient amount of lightat an appropriate wavelength to excite the selected tracer.

The embodiment of FIG. 1 further includes a 45° angle mirror 110 whichis positioned for making beam 102 a vertical beam prior to focussing thebeam onto the biosensor. It will be understood by those skilled that thenumber and arrangement of mirrors 104, 106, 108, 110 may be varied asnecessary to accommodate various space limitations, with the solerequirement being that a sufficient amount of light be directed tobiosensor 120.

Biosensor 120 has an optical substrate 122 with one end 124 positionedto receive light from beam 102. A focussing lens 126 is positionedbetween angle mirror 110 and end 124 of waveguide 122, for focussinglight from beam 102 onto end 124. Desirably, focussing lens 126 ismounted on an X-Y translation unit so that its position may be adjustedfor best focussing.

Light detection means indicated generally at 150 are positioned todetect fluorescent light emitted from biosensor 120. The emitted lightis reflective of the concentration of a selected analyte in a sample, asis better described subsequently in reference to FIGS. 2 and 7-10. Lightdetection means 150 includes a collection lens 152 positioned to collectthe emitted fluorescence in a direction substantially orthogonal to thedirection of propagation of light beam 102 through optical substrate122.

The distance 154 between collection lens 152 and optical substrate 122is selected as known to those skilled to maximize the collection oflight emitted from the region of evanescent light penetration. The lightcollected by collection lens 152 is then sent to detection means 150,which responds by outputting signals reflective of the level ofcollected fluorescence light.

Detection means 150 may be any type of photodetector useful to detectlight in the wavelength region spanning the wavelength range of theemitted fluorescence, as known in the art. In FIG. 1, detection means150 is a CCD (charge-coupled device) detector which produces a signallike that depicted in FIG. 4C. Means are provided to integrate thesignal function around each peak to determine the total collectedfluorescence from the sample.

Alternatively, detection means 150 may be a photomultiplier, asemiconductor photodiode, or an array of such detectors. In embodimentsother than a CCD, an array is generally preferable to a single detectorfor some purposes. With an array of small detectors, the user candetermine that the peak fluorescence is being detected and is notinadvertently missed due to misalignment of the collection and detectionoptics. However, in an embodiment for routine use such as in a testinglaboratory, and for which all the parameters of the assay have beenstandardized, the spectrograph may be replaced by a filter which passesonly wavelengths in the region of tracer fluorescence.

In contrast to the rod-shaped fiber optic waveguides typically found inimmunofluorescent assay devices, in the present invention opticalsubstrate 122 is of generally planar shape having two planar surfacesspaced by a width, as shown in FIG. 2. Optical substrate 122 may forexample be a square or rectangular glass microscope slide or coverslip,or the like. Materials for optical substrate 122 include glass,high-lead glass, quartz, optical plastic, and the like as are well-knownin the art.

For focussing light beam 102 onto the end of the planar substratewaveguide, it is preferred to replace the typical spherical lens with alens of approximately half-cylindrical shape, as better seen in FIGS. 3Cand 5A. The curved side of the lens need not be a strict half cylinder,but may have an ellipsoidal profile.

As is better seen in FIG. 2, optical substrate 122 is embodied as aplanar waveguide having at least one planar surface 200 spaced from asecond surface 201 by a width 202. At least surface 201 is disposed incontact with a sample solution 203. A plurality of capture molecules 204are immobilized on surface 201. The sample solution contains a pluralityof analyte molecules 210 of a selected analyte, and a plurality oftracer molecules 220. The capture molecules are chosen or constructed tobind to a binding moiety present on each of the analyte molecules 210.The tracer molecule 220 is chosen or constructed to emit fluorescentlight in response to stimulation by light of the appropriate wavelength.The level of fluorescence emitted by the tracer molecules 220 is ameasure of the amount of analyte bound to the capture molecule and isthereby reflective of the concentration of analyte molecules 210 in thesolution.

When light is being propagated in the waveguide 124 and internallyreflected at the surfaces 200, 201, an evanescent light field isproduced having an intensity curve 230 which drops off with distancefrom the surface 200, as diagrammed relative to a distance axis 232 andan intensity axis 234 (not to scale). It will be apparent that anexcitation zone 240 is the only region of the solution in which theevanescent light intensity is sufficient to excite a significant ordetectable fraction of tracer molecules 220 (not to scale). Tracermolecules 220 which are outside this zone will contribute little or noinduced fluorescence. Excitation zone 240 is typically between about1000 and 2000 Å in width.

Capture molecules 204 may be whole antibodies, antibody fragments suchas Fab' fragments, whole antigenic molecules or antigenic fragments, andoligopeptides which are antigenic and/or similar in 3-dimensionalconformation to an antibody-binding epitope.

In FIG. 2, a competition assay scheme is depicted (also termed adisplacement assay). However, with appropriate modifications to theassay scheme which will be apparent to the skilled person, alternateassay schemes such as sandwich assays may be performed with the presentapparatus.

The capture molecules 204 may be immobilized on the surface 202 by anymethod known in the art. However, in the preferred embodiment thecapture molecules are immobilized in a site-specific manner. As used inthis application, the term "site-specific" means that specific sites onthe capture molecules are involved in the coupling to the waveguide,rather than random sites as with typical prior art methods. ExamplesI-III detail methods for site-specific immobilization of capturemolecules to the surface of the optical substrate by means of aprotein-resistant coating on the substrate.

As previously stated, the intensity of evanescent light drops offrapidly with distance from the waveguide surface. Thus, only tracermolecules which are within an effective excitation range 240 (notnecessarily to scale) from the waveguide surface, will be excited by theevanescent light to emit fluorescence. The range 240 is generally about1000 to 2000 Å. This range is sufficient to ensure that essentially alltracer molecules 220 which are bound (directly or indirectly) to capturemolecules 204, will be detected, while the bulk of the tracer moleculeswhich remain free in solution are outside the effective excitationrange.

In a working embodiment of the apparatus of FIG. 1, measurements offluorescence are made by spectroscopy. For the examples involvingrhodamine-tagged molecules, light source 100 is an argon ion laser (aLEXEL Model 95-2) at an emission wavelength of 514 nm. Fluorescencedetection was done with a monochromator (SPEX Industries, Inc., Model1680C) and a charge-coupled device (abbreviated CCD) (Photometrics Ltd.Series 200). Alternatively, light source 100 can be any laser emittingat the wavelength desired for excitation of selected fluorescent dyes.Also, once an assay procedure has been validated and standardized, itmay not be necessary to measure the fluorescence spectrum or spatialdistribution of fluorescence. The detection means may be simplified inaccordance with the minimum requirements of the assay.

In another alternate embodiment, light source 100 is a laser diodeemitting in the red wavelength region of 600-700 nm, available fromHitachi. This laser diode provides about 5 milliwatts of power with apeak emission wavelength of about 670 nm. For an embodiment using alaser diode, it is necessary to use dyes such as cyanine dyes, whosefluorescence can be stimulated by excitation with wavelengths in the redspectral region. An example of such a dye is CY5, available fromBiological Detection Systems, Inc., Pittsburgh, Pa. (catalog no.A25000). The CY5 dye can be conjugated to the desired tracer molecule bythe manufacturer's instructions and/or with a kit available from BDS.The dyes and methods for conjugating are characterized in the paper bySouthwick, P. L., et al., titled "Cyanine Dye LabellingReagents--Carboxymethylindo-cyanine Succinimidyl Esters", Cytometry11:418-430 (1990).

In the experiments described herein and whose results are shown in FIGS.7-10, data acquisition and processing was accomplished using softwaresupplied with the Photometrics Series 200.

In the embodiment of FIG. 2, the immunoassay is a competition assay inwhich the tracer molecules 220 are constructed such that capturemolecules 204 will bind tracer molecules 220 in place of analytemolecules 210. Higher concentrations of analyte molecules 210 will causemost of the tracer molecules 220 to be displaced into the surroundingsolution from capture molecules 204, thus reducing the number of tracermolecules within excitation range 240 of the substrate 122. This reducedbinding of tracer molecules in turn reduces the amount of fluorescence.In contrast, lower concentrations of analyte molecules 210 will allowtracer molecules 220 to bind to capture molecules 204, and thus to beheld within the excitation range 240.

In the embodiment of FIG. 1, biosensor 120 is shown as a flow-throughcell, shown in greater detail in FIGS. 3A-B. A planar waveguide 302which may be for example a microscope slide or coverslip, is sandwichedbetween two plates 304, 306 which are held together by screw fittings308A, 308B. A gasket 320 is seated between waveguide 302 and plate 306.Gasket 320 is configured with two internal openings which, when gasket320 is securely sandwiched between plate 306 and waveguide 302, formreservoirs 322, 324. In reservoirs 322, 324, waveguide 302 constitutesone wall, plate 306 constitutes a second wall, and the inner edges 322A,324A of the gasket form the remaining walls. Although the reservoirs322, 324 are here shown to be rectangular in shape, other shapes couldbe used. Also, instead of two reservoirs as depicted in FIG. 3A, thegasket could have either just one opening or more than two, creatingcorresponding numbers of individual reservoirs.

Gasket 320 is preferably made of a semi-rigid material having an indexof refraction less than that of the waveguide material in the wavelengthrange of the exciting light. For best results, it is believed that theindex of refraction of the gasket material should be as low as possiblecompared to that of the waveguide. For a waveguide made of quartz orglass would typically be about 1.5, higher for high-lead glass. Atransparent (non-pigmented) silicon rubber (siloxane polymer) with anindex of refraction of 1.35-1.4 is a presently preferred material forgasket 320. TEFLON materials such as FEP or TEF (polytetrafluoroethyleneand --) often have indices of refraction of around 1.4-1.45, and mayalso be suitable. However, because TEFLON surfaces tend to adsorbprotein in a non-specific manner, silicon rubber is generally preferred.The Teflon-type materials, FEP or TEF, are also acceptable.

The lower plate 306 in FIG. 3B, has a pair of inlets 330, 332 and a pairof outlets 340, 342. These inlets and outlets are arranged so as topermit solutions to flow separately through the respective reservoirs322, 324. Desirably, the lower plate 306 may be made from aluminumalloy.

FIG. 3C shows the waveguide 302 in isolation from the remaining parts ofthe biosensor. Lens 126 is shown receiving and focussing light beam 102onto the waveguide. Desirably, the outer, surrounding edge 350 is coatedwith a reflective material, except for an uncoated region 352 at whichthe focussed light from lens 126 enters the waveguide (FIG. 3C). Arrows354 indicate reflection from the coated edges. In FIG. 3C, only one lensand one uncoated region are shown, however, for two or more channels,more portions of edge 350 may be left uncoated to allow light to enterthe waveguide (see for example FIG. 4A).

The reflective coating reflects back into the waveguide, light thatwould otherwise escape through the edge 303. The intensity of theevanescent light wave is thereby enhanced. Suitable reflective coatingmaterials include aluminum, silver, or the like, as known in the art.Alternatively, in place of a coating, reflectors could be positionedabout the edges to reflect escaping light back into the waveguide.

The design with at least two individual reservoirs has significantadvantages over a single reservoir embodiment for instances in which itis desirable to measure the test sample fluorescence simultaneously withfluorescence from a control region on the same waveguide. For example,the level of non-specific binding to the waveguide can be subtractedfrom the test sample fluorescence. Also, measurement changes due tofluctuations in intensity of the exciting light can be corrected for. Ina displacement assay, the "control" region could be the preloadedwaveguide with no analyte present in the sample, or with a known amountof analyte.

FIGS. 5A-5D depict an alternate embodiment of a biosensor useful withthe apparatus of FIG. 1. The biosensor indicated generally at 500 has anintegrally mounted or formed focussing lens 502 and waveguide 504arranged such that lens 502 focusses light onto the forward end 506 ofthe waveguide. Focussing lens 502 is configured and positioned to focusa light beam onto the receiving end 506 of the waveguide 504 (FIGS. 5A,5C). Side walls 511, 512, top and bottom walls 516, 517, and a removablysealing rear wall 518 enclose the space about the waveguide 504 tocreate reservoirs 520, 522.

The integral focussing lens 502 replaces focussing lens 126 in theapparatus of FIG. 1. In the working embodiment of FIGS. 5A-5D, thefocussing lens is molded as part of the waveguide holder 500 of anoptical plastic such as polystyrene, polycarbonate or the like.

Biosensor 500 also includes reservoirs 520, 522 best seen in FIGS. 5B,5C and 5D in which sample solutions can be disposed. Optionally, forsome applications it may be desirable to provide lengthwise ribs 530(FIG. 5D) along slot 504 which can define separate regions of thewaveguide surface.

FIG. 6 depicts an alternate multiwell biosensor similar to that of FIGS.5A-5C, except that a series of discrete wells 600, 602, 604, 606 areformed on the waveguide 504. The embodiment of FIG. 6 would be used in ahorizontal position, so that the wells 600, 602, 604, 606 need not becovered.

The biosensor including the lens may be formed by molding of a suitableoptical plastic. The holder may be pre-molded, and a silica-surfacewaveguide inserted subsequently with a refractive-index-matched adhesiveto secure it in place and seal it as needed to create separate channels.Alternatively, the waveguide holder may be molded with a silica-surfacewaveguide in place, thereby eliminating the need for the adhesive.

In still another embodiment, the waveguide could itself be formed of theoptical plastic and molded simultaneously with the holder. The lattertype construction is not suitable for use with excitation wavelengths of488 to 515 nm, because known optical plastics tend to emit fluorescencein this (the blue) wavelength region. However, an alternate embodimentof the apparatus using a laser diode as the light source emitting atwavelengths of 600 nm and above, would accommodate a plastic waveguide.

The following examples detail three methods for attaching the capturemolecules to the waveguide surface in a site-specific manner. Thegeneral scheme for reducing the level of non-specific binding is to coatthe waveguide with a protein-resistant material, and then immobilize theantibody to the coating. The scheme further includes derivatizing theprotein-resistant coating combined with site-specific modification ofthe antibody or other capture molecule to be immobilized, so as toprovide site-specific attachment of the capture molecule to the coating.Of the three examples presented, the procedures of Examples I and IIgave generally better results. At present, the avidin-biotin couplingmethod (Example II) is the most preferred. Using either coupling scheme,at least about 75% of the immobilized Fab' fragments were active, andthe levels of non-specific binding were typically no more than 1-2% ofthe specific binding. The modified PEG coating gave slightly higherlevels of non-specific binding, in the range of 5% to about 25%.

EXAMPLE I Preparation of Waveguide Surface--Hydrogel

A silica surface was prepared with a hydrogel coating comprised ofpolymethacryloyl hydrazide (abbreviated PMahy). Fused silica slides ofCO grade and thickness about 1 mm, available from ESCO, Inc., weresuitable as waveguides (optical substrates).

To graft the PMahy to the silica, the surface was derivatized withaldehyde groups. The derivatization was accomplished by silanizationwith 3-aminopropyltriethoxy silane (abbreviated APS) to add an aminofunctional group, followed by reaction with glutaraldehyde to producefree aldehyde groups. The PMahy was then be reacted with these aldehydegroups to form the hydrogel coating.

Antibodies could be coupled to this hydrogel in at least two ways. Inone method, the carbohydrate groups in the Fc antibody region areoxidized to aldehydes by treatment with sodium metaperiodate. However,few antigen-binding fragments contain carbohydrate moieties useful forthis purpose. Thus, a preferred method comprised modifying the pendanthydrazido groups of the hydrogel to a maleimido group by treatment withsuccinimidyl 4-(N-maleimido-methyl)cyclo-hexane-1-carboxylate(abbreviated SMCC; Pierce Chemicals). These maleimido groups can bereacted with the free thiol groups typically found in the C-terminalregion of Fab' fragments, thereby coupling the Fab' fragments to thehydrogel.

Polymethacryloylchloride (abbreviated PMaCl) was prepared by radicalpolymerization of methacryloyl chloride (abbreviated MaCl) in dioxaneunder an inert atmosphere, as described in Jantas et al., J. Polym.Sci., Part A: Polym. Chem. 27:475-485 (1989).

A reaction mixture containing 21.1. mole % of MaCl, 78.1 mole % dioxane,and 0.8 mole % AIBN (azobisisobutyronitrile), was allowed to react for24 hours at 60° C. with agitation. The PMaCl so produced remained insolution during the course of the reaction. The mixture was then dilutedwith twice the amount of dioxane used in the reaction and slowly addedto an excess of hydrazine hydrate, to achieve a volumetric ratio of 2:5for diluted PMaCl. The latter addition was carried out for about 30minutes in an ice bath under a nitrogen atmosphere. The resultingmixture was then stirred for about an hour at room temperature. Theproduct PMahy was purified by evaporation of dioxane and the remainingunreacted hydrazine hydrate, followed by washing in distilled water. Thewashed product was then dialyzed in a SpectraPor dialysis membranehaving a molecular weight cut-off of 3,500 daltons, to remove unreactedmonomer.

The polymer so prepared was shown to have a molecular weight of about26,000 as measured by gel permeation chromatography for thehydrochloride form. The concentration of polymer in solution in thehydrochloride form was estimated to vary between about 5% and 8% (w/v).It has been found that the polymer can be stored in aqueous solution at4° C. under a nitrogen atmosphere, for at least 5 months withoutundergoing a detrimental amount of spontaneous cross-linking.

Silica chips or glass or quartz microscope slides were cleaned withchromic acid, then treated with 5% APS/95% deionized water (v/v) forabout fifteen minutes at room temperature. The APS-treated surfaces wererinsed with deoionized water and absolute ethanol, and incubated in avacuum oven which had been flushed at least three times with nitrogen,at 120° C. for 1 hour. The resulting silanized surfaces were then soakedin 2.5% glutaraldehyde (E.M. grade from Polysciences) in 0.1Mcarbonate-bicarbonate buffer, pH 9.2, for two hours at room temperature.

Next, linear PMahy was reacted with the aldehyde groups on the treatedchips to create a cross-linked polymer film with many unreactedhydrazido groups in the chains. This was done by dipping the treatedchips in solutions of PMahy of between about 5% and 8% (w/v), pH 5.2, ata temperature between about room temperature and about 60° C., for atime sufficient to form a polymer film of thickness about 100 Å or less.The thickness of the hydrogel layer increases with time and temperatureof incubation in the solution. It was found that optimal conditions forpreparation of the film of 100 Å thickness or less, comprised incubatingin 5% (w/v) PMahy for 2 hours at room temperature (about 25° C.).

Next, the free hydrazido groups of the polymer film were modified bytreatment with SMCC to provide reactive maleimido groups on the ends ofthe polymer side chains. This was done by immersing the PMahy-coatedsubstrates in a solution of 0.19% (w/v) SMCC in dimethylformamide forabout 1 hour at 25° C.

Following derivatization with SMCC, the hydrogel-coated surfaces weretreated with a 1 mg/ml solution of Fab' fragments in phosphate buffer,ph 6.0, with 5 mM EDTA. The waveguide surface so prepared was shown toimmobilize IgG molecules at a surface density of about 1.4×10⁻¹²moles/cm². Also the surface was able to immobilize Fab' fragments attheir C-terminal thiol groups in a site-specific way. The thickness ofthe resulting polymer film was determined by ellipsometry to be about100 Å, as was desired. This film thickness is much less than typicalprevious polymeric films, which have thicknesses of 0.35 to 25 μm(microns). The above-described method of preparing the PMahy polymers issuperior to that described by von Kern et al. using polymethacryloylacidesters. Such esters suitable for reaction with hydrazine hydrate oftenhave a molecular weight of 80,000 daltons or more, from which it isdifficult to obtain a desirably thin film on the waveguide.

Finally, the Fab' fragments were coupled to the free maleimido groupspendant from the polymer-coated surface as follows. The preparedwaveguide surface was incubated for 24 hours at 4° C. in a solutioncontaining the Fab' fragments at a concentration of 1.5×10⁷ molar, in aphosphate buffer with 5 mM EDTA (pH 6.0).

EXAMPLE II Preparation of Waveguide Surface--Avidin-Biotin

This strategy was designed to exploit the very strong binding affinityof biotin for avidin (binding constant of around 10⁻¹⁵). An avidincoating was readily made by physical adsorption on a silica surface. TheFab' fragments were then conjugated with biotin to form biotin-Fab'conjugates, also referred to as biotinylated Fab' fragments or b-Fab'fragments. The biotin is coupled at specific location(s) on the Fab'fragments. The avidin coated surface is then treated with the b-Fab'fragments, so that the biotin binds to the avidin thereby immobilizingthe Fab' fragment to the surface in a site-specific manner.

In actual experiments, the procedure was as follows. Chromicacid-cleaned silica surfaces were immersed in a solution of 3×10⁻⁶ M(molar) avidin for about 3 hours at room temperature. The surfaces werethen washed several times in PBS to remove unadsorbed avidin.

Biotinylated Fab' conjugates were prepared from a solution of Fab'fragments in PBS (0.5-1 mg/ml), by addition of a sufficient amount of 4mM biotin-HPDP in dimethylformamide to provide a 20-fold molar excess ofbiotin-HPDP. This mixture was incubated for 90 minutes at roomtemperature, and biotinylated Fab' fragments (abbreviated b-Fab') werepurified by gel permeation chromatography with Sephadex G25 equilibratedin PBS.

An alternate method was used for biotinylating whole antibodies, inwhich biotin-LC-hydrazide was coupled to oxidized carbohydrate groups inthe Fc region of the antibody. Mab designated 9-40 (a murine monoclonalIgG₁ antibody that binds fluorescein), was oxidized by incubation at aconcentration of 1-2 mg/ml protein in 10 mM sodium periodate, 0.1Msodium acetate pH 5.5 for 20 minutes at about 0° C. Glycerol was thenadded to a final concentration of 15 mM to quench the reaction, and themixture incubated a further 5 minutes at 0° C. The oxidized Mab 9-40 waspurified by gel filtration chromatography on Sephadex G25 equilibratedwith 0.1M sodium acetate buffer pH 5.5, and then reacted with 5 mMbiotin-LC-hydrazide for 2 hours at room temperature with agitation.Unreacted biotin-LC-hydrazide was removed using a Sephadex G25 columnequilibrated in PBS.

Avidin-coated surfaces were immersed in a 1.5×10⁻⁷ M solution of b-Fab'fragments for about an hour at room temperature, followed by washingwith PBS to remove unbound b-Fab' fragments. Optionally, polyethyleneglycol (abbreviated PEG) was coupled to surfaces that were previouslycoated with the b-Fab' fragments, by immersion of the b-Fab'-coatedsurfaces in a solution of between about 5×10⁻⁸ and 1×10⁻⁷ M PEG. UnboundPEG was removed by washing in PBS.

EXAMPLE III Preparation of Waveguide Surface--PEG-Type

In this method, the terminal hydroxyl groups of polyethylene glycol(abbreviated PEG) were converted to primary amine or hydrazide groups byreaction with ethylenediamine (abbreviated ED) or hydrazine,respectively. The PEG molecules so modified were then coupled toAPS-glutaraldehyde activated silica surfaces.

Monofunctional (PEG M2000, M5000) or difunctional (PEG 3400, PEG 8000,PEG 18,500) of the indicated molecular weights in daltons, were reactedwith p-nitrophenyl chloroformate (abbreviated p-NPC; obtained fromAldrich Chemicals) in solution in benzene. The mixture was agitated atroom temperature for about 24 hours. Dry ethyl ether (less than 0.01%water, purchased from J.T. Baker Chemicals) was used to precipitatePEG-(o-NP)₂ from solution. The precipitate was vacuum-dried overnight.Between about 50% and about 100% of PEG molecules were converted by thistreatment to PEG-Onp, as determined by hydrolysis with 0.1N sodiumhydroxide to release the p-nitrophenol groups. The absorbance at 402 nmwas determined spectrophotometrically and a molar extinction coefficientof 18400M⁻¹ cm⁻¹ used to determine the amount of conversion. The levelof conversion depended somewhat on the molecular weight of the PEG ofMPEG.

PEG-(o-NP)₂ was then dissolved in ethylenediamine and agitated gentlyfor about 3 hours at room temperature. The PEG-(ED)₂ was thenprecipitated by addition of a sufficient amount of dry ethyl ether. Theyellow PEG-(ED)₂ solution was decolorized by addition of 1 drop of 12N(normal) hydrochloric acid, and the precipitation with ethyl etherrepeated twice more. The wet PEG-(ED)₂ was dried under vacuum overnight.Alternatively, in place of ethylenediamine, the PEG was derivatized withhydrazine to produce PEG-Hz₂.

The modified PEG was coupled to silanized-glutaraldehyde-treatedwaveguide surfaces prepared as described in Example I. A solution of 24milligrams (mg) of PEG-ED powder dissolved in 1.2 milliliters (ml) of0.15M PBS pH 7.4 or in the same volume of 11% potassium sulfate-sodiumacetate buffer at pH 5.2. The prepared waveguide surfaces were immersedin the PEG-ED solution and incubated at 60° C. for about 24 hours. Theprocedure using K₂ SO₄ -acetate buffer yielded a higher density of PEGmolecules attached to the surface than that using PBS buffer.

    __________________________________________________________________________    Summary of Solid-Phase Immunoassays using Silica Substrates                                        Absolute                                 Total  Non-specific                                               Relative                                                       Immobilized                                 Binding.sup.3                                        Binding.sup.4                                               Non-specific                                                       Antibody.sup.5                                                              Specific                           Coupling                                 (× 10.sup.-12)                                        (× 10.sup.-12)                                               Binding (× 10.sup.-12)                                                              Activity.sup.6    Surface.sup.1                 Antibody.sup.2                           Chemistry                                 (moles/cm.sup.2)                                        (moles/cm.sup.2)                                               (%)     (moles/cm.sup.2)                                                              (%)    __________________________________________________________________________    Hydrophobic Silica                 Heat Treated IgG                           Random                                 0.65   0.04   6.46    3.00   21.67    (DDS)    Hydrophobic Silica                 Acid Treated IgG                           Random                                 0.67   0.07   9.70    2.20   30.45    (DDS)    Hydrophobic Silica                 Fab'      Random                                 0.37   0.25   69.00   1.30   28.46    (DDS)        Fragment    Silica/APS/GLU                 Acid Treated                           Random                                 0.55   0.21   38.18   3.75   14.67                 IgG    Silica/APS/GLU/PEG                 Oxidized IgG                           Specific                                 0.56   0.11   19.64   2.06   27.18    Silica/APS/GLU/PEG                 Oxidized IgG                           Random                                 0.41   0.10   24.39   1.44   28.47    Silica/Avidin                 Biotin-IgG                           Specific                                 0.72   0.02   2.92    0.94   76.60    Silica/Avidin                 Biotin-Fab                           Specific                                 0.84   0.02   2.62    1.10   76.36    Silica/Avidin                 Biotin-Fab                           Specific                                 0.80   0.02   1.88    1.10   72.73    (with biotin-PEG)    Silica/Hydrogel                 Oxidized IgG                           Specific                                 0.17   0,01   6.88    7.95   2.14    (preswollen)    Silica/Hydrogel                 Fab'      Specific                                 1.51   0.03   2.03    2.76   54.71    (preswollen) Fragment    __________________________________________________________________________     .sup.1 Abbreviations: DDS -- dichlorodimethylsilane; APS --     aminopropysilane, GLU -- glutaraldehyde; PEG -- polyethylene glycol (3400     MW); BSA -- bovine serum albumin; IgG -- intact immunoglobulin G; Fab' --     antigen binding fragment with reactive thiol group; ND -- not determined     .sup.2 All immunoassays were performed with an IgG, monoclonal antibody     (940) which binds fluorescein.     .sup.3 Amount of .sup.125 IFluorescein-BSA which bound to silica     substrate.     .sup.4 Amount of .sup.125 IBS4 which bound to silica substrate.     .sup.5 Amount of .sup.125 I9-40 immobilized on silica substrate.     .sup.6 Percent of immobilized active sites which bound antigen molecules.

                                      TABLE II    __________________________________________________________________________    Summary of Solid Phase Immunoassays Using Silica Substrates Covered with    Hydrogel with Maleimido Reactive Groups                                        Absolute                                               Relative                                                     Absolute                                                            Relative                      Total hCG                            Total hCG   Non-specific                                               Non-  Non-specific                                                            Non-specific               Immobilized                      Binding in                            Binding in  Binding in                                               specific                                                     Binding in                                                            Binding in          Binding               Antibody                      5 min 60 min                                  Antibody                                        5 min (BSA)                                               Binding in                                                     min (BSA)                                                            60 min          Constant               (× 10.sup.-12                      (× 10.sup.-12                            (× 10.sup.-12                                  Activity                                        (× 10.sup.-12                                               5 min (× 10.sup.-12                                                            (BSA)    Antibody          pK.sub.0               mol/cm.sup.2)                      mol/cm.sup.2)                            mol/cm.sup.2)                                  (%)   mol/cm.sup.2)                                               (BSA) (%)                                                     mol/cm.sup.2)                                                            (%)    __________________________________________________________________________    Fab' from          8.85 1.39 ± 0.07                      0.62 ± 0.03                            0.81 ± 0.03                                  58.3 ± 0.8                                        <0.01  <0.97 <0.02  <2.47    Anti-    hCG-A    Fab' from          7.89 1.29 ± 0.06                      0.51 ± 0.02                            0.67 ± 0.03                                  51.9 ± 0.1                                        0.02 ± 0.01                                               3.85 ± 1.81                                                     0.04 ± 0.01                                                            5.91 ± 1.22    Anti-    hCG-B    Fab' from          8.70 0.74 ± 0.04                      0.18 ± 0.01                            0.41 ± 0.02                                  55.4 ± 0.3                                        <0.01  <6.22 0.03 ± 0.01                                                            7.21 ± 2.09    Anti-    hCG-C    Fab' from          8.00 1.45 ± 0.06                      0.58 ± 0.02                            0.75 ± 0.03                                  51.4 ± 0.3                                        <0.01  <1.20 <0.02  <3.21    Anti-    hCG-D    Fab' from          --   2.50 ± 0.10                      0.04 ± 0.01                            0.06 ± 0.02                                  2.4 ± 0.7                                        --     --    --     --    Mouse IgG    __________________________________________________________________________

                                      TABLE III    __________________________________________________________________________    Summary of Solid Phase Immunoassay Using Silica Substrates with Adsorbed    Avidin    and Biotinylated Fab' Fragmebts                               Absolute                               Non-specific                                      Relative Non-           Immobilized                  Total hCG    Binding                                      specific           Antibody                  Binding                         Specific                               (BSA)  Binding           (× 10.sup.-12                  (× 10.sup.-12                         Activity                               (× 10.sup.-12                                      (BSA)    Antibody           mol/cm.sup.2)                  mol/cm.sup.2)                         (%)   mol/cm.sup.2)                                      (%)    __________________________________________________________________________    Fab' from           1.19 ± 0.02                  1.22 ± 0.01                         100 ± 5                               0.05 ± 0.02                                      4.20 ± 0.02    Anti-hCG-A    Fab' from           1.40 ± 0.05                  1.38 ± 0.07                         98 ± 9                               0.05 ± 0.01                                      3.57 ± 0.02    Anti-hCG-B    Fab' from           2.24 ± 0.02                  1.10 ± 0.03                         49 ± 3                               0.05 ± 0.02                                      2.32 ± 0.01    Anti-hCG-C    Fab' from           1.59 ± 0.02                  1.24 ± 0.01                         78 ± 2                               0.05 ± 0.005                                      3.14 ± 0.01    Anti-hCG-D    Fab' from           1.25 ± 0.02                  0.03 ± 0.003                         2.4 ± 0.05                               0.09 ± 0.03                                      7.20 ± 0.03    Mouse IgG    __________________________________________________________________________

Antibodies or other binding proteins were immobilized to the PEG-coatedwaveguides as follows. A solution of about 3 mg/ml of antibody wasdissolved in 0.15M sodium acetate buffer, pH 5.2. A solution ofequivalent weight of 50 mM sodium metaperiodate (NaIO₄) was then added,and the reactants were agitated at room temperature for about an hour.Unreacted sodium metaperiodate was removed by passing the reactionmixture through a desalting column (type PD-10 from Pharmacia), whichhad been pre-equilibrated with the sodium acetate buffer.

The PEG-coated waveguides were then incubated with the oxidized antibodysolution in the sodium acetate buffer, pH 5.2, for 3 days at 4° C., thenrinsed to remove unbound antibody.

The levels of non-specific absorption of antigen on waveguides preparedby this method were around 25-27%, which is considerably lower than thatobserved with prior art methods.

For the procedures detailed in Examples I-III and for which a comparisonof results is given in Table I, the Fab' fragments used were derivedfrom a murine anti-human chorionic gonadotrophin (anti-hCG) monoclonalIgG antibody. The parent monoclonal antibody was purified as describedby van Erp et al. J. Immunol. Methods, 140:235-241 (1991). This mouseantibody, termed anti-hCG-A, is directed against a portion of theβ-subunit of hCG (provided by Organon-Teknika of Boxtel, Netherlands).The whole monoclonal antibody anti-hCG-A was used in the experimentswhose results are depicted in FIGS. 7-10.

F(ab')₂ fragments were produced by digestion with pepsin using theprocedure described by Grey and Kunkel, "H Chain subgroups of myelomaproteins and normal 7S globulin," J. Exp. Med. 120:253-266, 1964.Following digestion, F(ab')2 fragments were reduced to Fab' fragmentsusing dithiothrietol (DTT). Specifically, 33 mg of purified antibody and1 mg pepsin (Sigma) were dissolved in 0.1M sodium acetate buffer (pH4.2) and the digestion was carried out at 37° C. for 16 hours. Thedigestion was terminated by adjusting the pH of the reaction mixture to8.0 with 2M tris base. The F(ab')₂ fraction was separated by gelpermeation chromatography (Superdex Hiload, Pharmacia) usingphosphate-buffered saline (PBS), pH 7.7, as eluent. Fab' fragments wereprepared by reducing the F(ab')₂ fragments (1 mg/ml) with 1.75 mM DTTand 3.5 mM ethylenediamine tetraacetate (EDTA) in 0.17M tris buffer (pH7.4) for 45 minutes at room temperature. After reduction, excess DTT wasremoved by gel permeation chromatography using a Sephadex G-25 column(Pharmacia) equilibrated in 0.1M sodium phosphate buffer (pH 6.0)containing 5 mM EDTA.

FIGS. 7A, 7B and 8 are charts depicting fluorescence intensity dataobtained using two alternate formats for performing a fluorescenceimmunoassay to detect an antibody. In these experiments, the detectionof antibodies to human chorionic gonadotrophin (abbreviated hCG) wasused as a model to determine which format provided the greatestsensitivity. It will be evident that the methods described could beadapted to the detection of any desired antibody in biological fluidssuch as plasma or serum, for example the detection of antibodies toproteins of viral and bacterial pathogens, depending only on obtainingthe necessary antigen for use as the capture molecule.

For purposes of the tests shown in FIGS. 7 and 8, the antibody to bedetected (the analyte) was chosen to be a monoclonal antibody to an hCGantigen designated hCG-A. The data of FIG. 7A were obtained with wholehCG molecules serving as the capture molecules (the antigen or analytebinding molecule) in the assay. The data of FIG. 7B were obtained usinga hexapeptide constructed to selectively bind the hCG-A antigen, as thecapture molecules. In both experiments, the tracer was a goat anti-mouseIgG labelled with tetramethylrhodamine (abbreviated TMR). For both assayformats, the capture molecule was biotinylated as described in ExampleII and immobilized on an avidin-coated silica substrate. The testantibody, anti-hCG A, was premixed with the tracer (goat anti-mouseIgG-TMR) in the test solution.

As will be understood by those in the art for a sandwichfluoroimmunoassay, the anti-hCG A antibody bound to the immobilizedcapture molecule, and the goat-anti-mouse igG-TMR tracer in turn boundto the mouse anti-hCG A antibody. In this way a fluorescent sandwichformed on the substrate surface with the TMR-portion of the tracermolecule being held within the region of evanescent excitation.

The immunoassays were performed using an interfacial fluorometerconstructed at the University of Utah. Silica waveguides with theappropriate respective immobilized antigens were placed in thedual-channel flow-cell of FIG. 3. The two channels were used for sampleand reference measurements, as described with respect to FIGS. 4A-C. Thelight source was the 514.5 nm emission of an air-cooled argon-ion laser.The laser beam was split into two parallel beams, which were focusedwith lenses into the two channels of the waveguide. Fluorescenceemission was recorded from 520 to 620 nm using a momochromator connectedto a computer-controlled CCD camera. The fluorescence spectrum wasintegrated from 560 to 600 nm to improve the signal-to-noise ratio. Thefollowing protocol was used in all experiments. Different concentrationsof anti-hCG A were premixed with the tracer antibody (concentrationfixed at 10⁻⁸ M) and injected into the sample channel. A 10⁻⁸ Mconcentration of tracer antibody was also injected into the referencechannel as a control. The fluorescence intensity of the sample channelwas plotted vs. anti-hCG A concentration and the fluorescence intensityof the reference channel was also plotted on the same set of axes (thisis really a plot of the non-specific binding of the tracer antibody vs.time, since no anti-hCG A was injected into the reference channel).FIGS. 7A and 7B show the results for the sandwich binding of anti-hCG Ato the immobilized hCG and the oligopeptide, respectively. FIG. 8 showsthe corresponding fluorescence enhancements for both cases. The datafrom FIGS. 7A and 7B were normalized for background fluorescence andreplotted as fluorescence enhancement (F_(sample) /Freference) versuslog analyte concentration. The response curve was similar for both ofthe immobilized antigens (whole hCG and oligopeptide antigen) over arange of antibody concentrations from 10⁻¹³ M to 10⁻¹⁰ M. However, wholehCG gave better precision.

It is also evident from FIGS. 7A, 7B and 8 seen that analyte levels(anti-hCG A) as low as 10⁻¹³ molar were detectable with the assay. In afurther embodiment, the tracer antibody concentration is reduced to10⁻¹⁰ M or less. This is expected to reduce background fluorescence dueto non-specific adsorption of the tracer antibody and thereby furtherimprove the sensitivity to 10⁻¹⁴ M or better.

FIG. 9 depicts data obtained using an antibody as the capture moleculeto detect an antigen, in a sandwich-type assay. As mentioned previously,two different antibodies are employed in a sandwich immunoassay--animmobilized capture antibody and a labelled tracer antibody in solution.Since the capture antibody and the tracer antibody must bind to distinctregions of the antigen, two different monoclonal antibodies which bindto different epitopes on the antigen are typically used in such assays.In addition to the anti-hCG-A, three other monoclonal anti-hCGantibodies (anti-hCG-B, anti-hCG-C and anti-hCG-D, respectively) wereobtained from Organon Teknika which bound to different epitopes than didanti-hCG-A. Since only anti-hCG A (the others also bind to certainhormones related to hCG), only six of the twelve possible pairwisecombinations of antibodies provide strict selectivity for hCG.

FIGS. 9A-F depict results obtained with different pairwise combinations,with Fab' fragments from anti-hCG A (Fab'-A) and immobilized towaveguides using the avidin-biotin coupling chemistry. Fab' fragmentsprepared from anti-hCG B, anti-hCG C and anti-hCG D were labeled withtetramethylrhodamine for use as tracer antibodies (designated Fab'-B,Fab'-C and Fab'-D, respectively). FIGS. 9A and 9B show results withFab'-B as the tracer molecule. FIGS. 9C, 9D show results obtained usingFab'-C as the tracer molecule. FIGS. 9E, 9F show results obtained usingFab'-D as the tracer molecule. Presently, Fab'-B and Fab'-C arepreferred for use as tracers in an hCG assay. The format using Fab'-A asthe capture antibody was generally superior in sensitivity to theconverse protocol, that is, to using Fab'-A as the tracer molecule andFab'-B, -C or -D as the capture molecule.

FIGS. 10A-D show data obtained from a competition or displacement assay.Fab'-A fragments were immobilized to waveguides using either theavidin-biotin chemistry (FIGS. 10A, 10B) or the hydrogel couplingchemistry (FIGS. 10C, 10D). The immobilized Fab'-A fragments werepreloaded with the tracer oligopeptide at a concentration of 10⁻⁸ M.Increasing concentrations of hCG were added to one channel of the flowcell (sample) and PBS buffer was added to the other (reference). Foreach coupling chemistry, the raw fluorescence intensities of the sampleand reference channels are shown in the panels on the left (10A & 10C)and the percent of full-scale fluorescence (in the absence of hCG) areshown in the panels on the right (10B & 10D). The latter values werenormalized for the change in reference fluorescence. Standard errorswere plotted for all data points, but in some cases were smaller thanthe plot marks.

In some experiments for the purpose of characterizing the apparatus andassay system, fluorescein-BSA (BSA=bovine serum albumen) conjugates withan epitope density of nine were the analyte and the capture moleculeswere anti-BSA Fab' fragments. It was found that non-specific binding tothe avidin-coated waveguide was acceptably low for antigen (analytemolecule) concentrations of less than about 10⁻⁵ M, without a wash step.

At present the sandwich immunoassay is preferred for several reasons.First, detection of concentrations down to at least 0.1 picomolar can bedemonstrated, as compared to picomolar concentrations for thecompetitive assay. Also, the instant sandwich immunoassay was capable ofdetecting concentrations ranging over five logs--from 10⁻⁸ M to 10⁻¹³ M.Thus, a single assay formulation using the sandwich procedure couldserve for a variety of applications where different detection limits arerequired.

While the preceding experimental examples and results were obtainedusing hCG antigen--anti-hCG antibody and fluorescein-anti-fluoresceinantibody systems, it will be understood by those skilled that theapparatus and the biosensor, as well as the site-specificwaveguide-coupling methods and assay formats, all are applicable toassays for any antigen or antibody for which the requisite reagents suchas appropriate capture molecules can be obtained, without undueexperimentation. It will further be understood that whiletetra-rhodamine, fluorescein, and cyanine dyes are specificallymentioned as useful for labelling of tracer molecules, the apparatus andmethods can also be useful with other fluorescent dyes capable of beingconjugated to the desired tracer molecule.

It will further be recognized that various modifications andsubstitutions may be made to the apparatus and the biosensor asdescribed herein, without departing from the concept and spirit of theinvention.

We claim:
 1. A fluorescence immunoassay, comprising the stepsof:providing a solid substrate formed of a material selected from thegroup consisting of silica-based materials constructed for propagationof light by total internal reflectance, and having a surface with aplurality of capture molecules indirectly immobilized thereon via acoating comprising 3-aminopropyltriethoxy silane bonded to saidsubstrate and a plurality of polymerized hydrophilic polymer chainsselected from the group consisting of hydrogel formed ofpolymethacryloyl and polyethyleneglycol, said hydrophilic polymer chainscoupled to said 3-aminopropyltriethoxy silane, said capture moleculeshaving a binding site which selectively binds a selected analyte;providing a light source operable to emit a light beam in a desiredwavelength range and positioned to send light into said substrate;providing detection means operably disposed for detecting fluorescenceemitted from the solid substrate; providing a sample comprising a bufferand a plurality of molecules of a selected analyte; providing aplurality of tracer molecules which are operable to emit fluorescence inresponse to stimulation by light from the light source, said bindingsites on said capture molecules also selectively bind to the tracermolecules; combining the sample with the tracer molecules to produce atest solution; placing the test solution in contact with the solidsubstrate while operating said light source to direct light into thesolid substrate; selectively detecting fluorescent light emitted frombound tracer molecules; and measuring the level fluorescence emitted todetermine the presence of analyte.
 2. The fluorescence immunoassay ofclaim 1, wherein said step of providing a solid substrate having capturemolecules immobilized thereon includes the steps of:coating thesubstrate surface with the 3-aminopropyltriethoxy silane to produce afirst layer; applying a second layer to said first layer, said secondlayer selected from the group consisting of: PEG polymers modified tocovalently couple to said first layer, said PEG polymers further havingan attached biotin moiety, and a hydrogel composed of polymethacryloylpolymers; and modifying said capture molecules to produce activatedcapture molecules constructed to bind to the coating; and treating thecoating with the activated capture molecules under conditions to causethe activated capture molecules to couple to the coating.
 3. The assayof claim 2, wherein said coating inhibits non-specific binding to lessthan about 10% of the specific binding of the tracer molecules to thesubstrate.
 4. The assay of claim 2, wherein said second layer provides aplurality of free maleimido groups.
 5. A fluorescence immunoassay,comprising the steps of:providing a solid substrate formed fromsilica-based materials constructed for propagation of light by totalinternal reflectance, and having a surface with a plurality of capturemolecules indirectly immobilized thereon via a coating comprising3-aminopropyltriethoxy silane bonded to said substrate and a pluralityof polymerized hydrophilic polymer chains selected from the groupconsisting of biotinylated polyethylene glycol of molecular weightbetween about 2000 and 5000, PEG polymers of molecular weight betweenabout 2000 and about 5000, and polymethacryloyl polymers of molecularweight between about 2000 and about 5000 coupled to said3-aminopropyltriethoxy silane, said capture molecules having a bindingsite which selectively binds a selected analyte; providing a lightsource operable to emit a light beam in a desired wavelength range andpositioned to send light into said substrate; providing detection meansoperably disposed for detecting fluorescence emitted from the solidsubstrate; providing a sample comprising a buffer and a plurality ofmolecules of a selected analyte; providing a plurality of tracermolecules which are operable to emit fluorescence in response tostimulation by light from the light source, said binding sites on saidcapture molecules also selectively bind to the tracer molecules;combining the sample with the tracer molecules to produce a testsolution; placing the test solution in contact with the solid substratewhile operating said light source to direct light into the solidsubstrate; selectively detecting fluorescent light emitted from boundtracer molecules; and measuring the level fluorescence emitted todetermine the presence of analyte.
 6. A biosensor for use with afluorescence immunoassay system having a light source producing a lightbeam, means for directing the light beam to irradiate a biosensor, anddetection means for detecting a tracer light signal reflective of anamount of analyte in a test sample in contact with the biosensor, thebiosensor comprising:reservoir means for holding one or more testsolutions, comprising at least one reservoir having walls; an opticalsubstrate constructed for propagation of light by total internalreflectance, and constituting one of said walls; a coating comprising3-aminopropyltriethoxy silane bonded to or avidin adsorbed on saidoptical substrate and a plurality of polymerized hydrophilic polymerchains selected from the group consisting of biotinylated polyethyleneglycol, PEG polymers, and polymethacryloyl polymers, each of saidpolymerized hydrophilic polymer chains carrying a covalent bindingmoiety constructed to react with a selected complementary moiety; and aplurality of analyte-binding molecules constructed to specifically bindan analyte, and including said complementary moiety and attached therebyto said chains; said coating constructed to limit nonspecific adhesionof protein thereto to less than about 10% of the specific binding of theanalyte to said analyte-binding molecule.
 7. The biosensor of claim 6,wherein said coating is selected from the group consisting of: hydrogelformed of polymethacryloyl polymers of molecular weight between about2000 and about 5000, polyethyleneglycol of molecular weight betweenabout 2000 and about 5000, and biotinylated polyethyleneglycol.
 8. Thebiosensor of claim 6, wherein said polymerized hydrophilic polymerchains consist of polyethylene glycol and polymethacryloyl polymers. 9.A kit useful for a solid state immunoassay, comprising an opticalsubstrate constructed for propagation of light by total internalreflectance, and formed of a material selected from the group consistingof: silica-based optical materials including glass and quartz; saidsubstrate having a surface coated with a coating comprising3-aminopropyltriethoxy silane adsorbed on said surface and a pluralityof hydrophilic polymers selected from the group consisting ofpolymethacryloyl polymers, polyethyleneglycol polymers, and biotinylatedpolyethyleneglycol, said hydrophilic polymers covalently bound to said3-aminopropyltriethoxy silane; and a plurality of capture moleculescoupled to said coating, said capture molecules each being constructedto specifically bind an analyte.
 10. The kit of claim 9, wherein saidnon-specific binding level is less than about 2% of said specificbinding level.
 11. The kit of claim 9, further including at least onetracer molecule constructed to bind with specificity to the analyte, andhaving a covalently-coupled fluorescent moiety.
 12. The kit of claim 11,wherein said capture molecule and said tracer molecule are both derivedfrom a member of an antibody-antigen pair, and the other member of saidantibody-antigen pair is said analyte.
 13. The kit of claim 9, whereinsaid capture molecule is a Fab' fragment formed by treating a wholeantibody with trypsin to produce an antibody fragment cleaved at acleavage site, and treating said cleaved antibody with a reducing agentto form a reactive thiol group at said cleavage site.
 14. The kit ofclaim 7, wherein said capture molecule has a thiol group reactivelydisposed thereon, wherein said capture molecule is covalently coupled tosaid coating via said thiol group and at least about 70% of said capturemolecules have analyte capture sites which are available for bindingsaid analyte, and wherein said substrate has a plurality of capturemolecules immobilized thereon.
 15. The kit of claim 9, wherein saidcoating comprises two layers, said first layer being applied directly tosaid surface and being selected from the group consisting of:aminopropyltriethoxysilane treated with glutaraldehyde, and avidin; andwherein said second layer is selected from the group consisting of:biotinylated PEG, PEG polymers of molecular weight between about 2000and about 5000, and polymethacryloyl polymers of molecular weightbetween about 2000 and about
 5000. 16. The kit of claim 9, wherein saidoptical substrate has opposing ends, and further including a lensintegrally adapted to said optical substrate at one of said ends, andconstructed to focus a light beam into said optical substrate forpropagation therein.
 17. The kit of claim 9, wherein said coatingcomprises two layers, said first layer being applied directly to saidsurface and being selected from the group consisting of:aminopropyltriethoxysilane and avidin; and said second layer beingapplied on said first layer and being selected from the group consistingof: biotinylated polyethylene glycol, PEG polymers of molecular weightbetween about 2000 and about 5000, and polymethacryloyl polymers.
 18. Akit useful for a solid state immunoassay, comprising a substrate elementconstructed for propagation of light by total internal reflectance; saidsubstrate element having a surface coated with a coating comprising3-aminopropyltriethoxy silane bonded to or avidin adsorbed on saidoptical substrate and a plurality of polymerized hydrophilic polymerchains selected from the group consisting of biotinylated polyethyleneglycol, polyethylene glycol polymers, and polymethacryloyl polymers;said polymerized hydrophilic polymer chains each carrying a covalentbinding moiety constructed to react with a selected complementarymoiety; and a plurality of capture molecules each constructed tospecifically bind an analyte and each having a single said complementarymoiety, wherein at least about 70% of said capture molecules haveanalyte capture sites which are available for binding the analyte, andwherein said capture molecules are covalently coupled to said coatingvia said complementary moiety and immobilized to said surface.
 19. Thekit of claim 18, wherein said polymerized hydrophilic polymer chains areselected from the group consisting of: polymethacryloyl polymers,avidin, and polyethyleneglycol polymers.
 20. The kit of claim 9, whereinsaid capture molecule is a Fab' 'fragment formed by treating a wholeantibody with trypsin to produce an antibody fragment cleaved at acleavage site, and treating said cleaved antibody with a reducing agentto form a reactive thiol group at said cleavage site.
 21. The kit ofclaim 18, wherein said capture molecule has a biotin moiety coupledthereto.
 22. A substrate element for use in a biochemical binding assayof the type in which an analyte is detected by its mediation of thespecific binding of a tracer molecule to a surface on which a capturemolecule which specifically binds the analyte is immobilized, preparedby the steps of:providing a substrate element formed of a silica-basedoptical material; reacting 3-aminopropyltriethoxy silane with a surfaceof said substrate element; coupling a plurality of polymerizedhydrophilic polymer chains selected from the group consisting of ahydrogel formed of polymethacryloyl and polyethylene glycol to said3-aminopropyltriethoxy silane; and coupling capture molecules whichspecifically bind to said analyte to said plurality of hydrophilicpolymer chains, wherein said substrate element provides a level ofnon-specific binding of the tracer molecule which is less than about 10%of the level of non-specific binding of the tracer molecule.
 23. Thebiosensor of claim 22, wherein said coating is aminopropylsilane oravidin.
 24. An apparatus for performance of a biochemical binding assay,comprisinga light source providing a light beam in a desired wavelengthrange; a biosensor includinga reservoir constructed for containing asample solution to be tested for presence of an analyte, a waveguidesubstrate constructed for propagation of light by total internalreflectance, and having at least one surface disposed within saidreservoir in contact with said sample solution, a reagent reacted tosaid at least one surface of said waveguide substrate, said reagentcreating a grafting surface, a coating comprising polymerizedhydrophilic polymer chains selected from the group consisting ofpolyethylene glycol polymers, and polymethacryloyl polymers, saidhydrophilic polymer chains being attached to extend from said waveguidesubstrate via said reagent, and a plurality of capture moleculesimmobilized to said at least one surface via said coating, said capturemolecules being constructed to specifically bind said analyte or atracer molecule; channeling means operably associated with said lightsource and said biosensor for directing light from said light source toirradiate said waveguide substrate; and detection means operablydisposed for detecting a light signal resulting from bound tracermolecules on said at least one surface of said waveguide substrate. 25.The apparatus of claim 24, wherein said polymer chains further havecovalently attaching functional groups attached thereto, and saidcapture molecules are covalently coupled to said covalently attachingfunctional groups.
 26. The apparatus of claim 25, wherein said surfacehas a plurality of capture molecules immobilized thereon, and wherein atleast about 70% of said capture molecules have analyte capture siteswhich are available for binding said analyte.
 27. The apparatus of claim25, wherein said covalently attaching functional group is a maleimidogroup.
 28. The apparatus of claim 27, wherein said capture molecule is aFab' fragment formed by treating a whole antibody with trypsin toproduce an antibody fragment cleaved at a cleavage site, and treatingsaid cleaved antibody with a reducing agent to form a reactive thiolgroup at said cleavage site.
 29. An optical substrate for use in abiochemical binding assay of the type is which an analyte is detected byits mediation of the specific binding of a tracer molecule to a surfaceon which a capture molecule which specifically binds the analyte isimmobilized, comprising:an optical element formed of an optical plasticor a silica-based optical material; a reagent reacted to said opticalelement; a coating attached to said optical element via said reagent andcomprising a plurality of polymerized hydrophilic polymer chainsselected from the group consisting of biotinylated polyethylene glycol,polyethylene glycol polymers, and polymethacryloyl polymers, each ofsaid chains carrying a covalent binding moiety constructed to react witha selected complementary moiety selected from the group consisting of athiol group and an oxidized carbohydrate moiety; and a plurality ofanalyte-binding molecules each constructed to specifically bind ananalyte, and including said complementary moiety and attached thereby tosaid chains.
 30. The optical substrate of claim 29, wherein at leastabout 70% of said capture molecules are reactively available for bindingsaid analyte.
 31. The optical substrate of claim 29, wherein saidoxidized carbohydrate moiety is formed by treating a whole antibody withsodium metaperiodate.